Telomere-binding Protein TRF2 Binds to and Stimulates the Werner and Bloom Syndrome Helicases

Werner syndrome is a human premature aging disorder displaying cellular defects associated with telomere(definition) maintenance including genomic instability, premature senescence(definition), and accelerated telomere erosion. The yeast homologue of the Werner protein (WRN), Sgs1, is required for recombination-mediated lengthening of telomeres in telomerase-deficient cells. In human cells, we report that WRN co-localizes and physically interacts with the critical telomere maintenance protein TRF2. This interaction is mediated by the RecQ conserved C-terminal region of WRN. In vitro, TRF2 demonstrates high affinity for WRN and for another RecQ family member, the Bloom syndrome protein (BLM). TRF2 interaction with either WRN or BLM results in a notable stimulation of their helicase activities. Furthermore, the WRN and BLM helicases, partnered with replication protein A, actively unwind long telomeric duplex regions that are pre-bound by TRF2. These results suggest that TRF2 functions with WRN, and possibly BLM, in a common pathway at telomeric ends. Werner syndrome is a human premature aging disorder displaying cellular defects associated with telomere maintenance including genomic instability, premature senescence, and accelerated telomere erosion. The yeast homologue of the Werner protein (WRN), Sgs1, is required for recombination-mediated lengthening of telomeres in telomerase-deficient cells. In human cells, we report that WRN co-localizes and physically interacts with the critical telomere maintenance protein TRF2. This interaction is mediated by the RecQ conserved C-terminal region of WRN. In vitro, TRF2 demonstrates high affinity for WRN and for another RecQ family member, the Bloom syndrome protein (BLM). TRF2 interaction with either WRN or BLM results in a notable stimulation of their helicase activities. Furthermore, the WRN and BLM helicases, partnered with replication protein A, actively unwind long telomeric duplex regions that are pre-bound by TRF2. These results suggest that TRF2 functions with WRN, and possibly BLM, in a common pathway at telomeric ends. Defects in members of the RecQ family of DNA helicases are responsible for three distinct human disorders: Werner syndrome (WS) 1The abbreviations used are: WS, Werner syndrome; WRN, Werner protein; TRF2, TTAGGG repeat binding factor 2; BLM, Bloom protein; RPA, replication protein A; ALT, alternative lengthening of telomeres; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GST, glutathione S-transferase; NE, nuclear extracts; ELISA, enzyme-linked immunosorbent assay; EGFP, enhanced green fluorescent protein; RQC, conserved RecQ C-terminal region; PML, promyelocytic leukemia. (1Matsumoto T. Shimamoto A. Goto M. Furuichi Y. Nat. Genet. 1997; 16: 335-336Google Scholar), Bloom syndrome (2Ellis N.A. German J. Hum. Mol. Genet. 1996; 5: 1457-1463Google Scholar), and Rothmund-Thomson syndrome (3Kitao S. Ohsugi I. Ichikawa K. Goto M. Furuichi Y. Shimamoto A. Genomics. 1998; 54: 443-452Google Scholar). All three disorders exhibit a predisposition to cancer and increased genomic instability (4Mohaghegh P. Hickson I.D. Hum. Mol. Genet. 2001; 10: 741-746Google Scholar). WS is of particular interest in aging research, because WS patients prematurely display aging features including gray hair, cataracts, osteoporosis, atherosclerosis, and diabetes mellitus (type II) (5Chakraverty R.K. Hickson I.D. Bioessays. 1999; 21: 286-294Google Scholar). Cells from WS patients show elevated levels of DNA deletions, translocations, and chromosomal breaks (6Fukuchi K. Martin G.M. Monnat R.J. Proc. Nat. Acad. Sci., U. S. A. 1989; 86: 5893-5897Google Scholar, 7Stefanini M. Scappaticci S. Lagomarsini P. Borroni G. Berardesca E. Nuzzo F. 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The status of the WRN protein in the cell influences not only genome integrity but also the cellular life span. The state of telomeres in the cell is also an important determinant of cellular life span. Telomeres cap and protect chromosome ends, and the consequences of telomere dysfunction include replicative senescence, apoptosis, and/or genomic instability (reviewed in Ref. 14Campisi J. Kim S. Lim C.S. Rubio M. Exp. Gerontol. 2001; 36: 1619-1637Google Scholar). Telomere dysfunction results from direct damage, defects in telomere maintenance proteins, and a progressive decline in telomere lengths that occurs with each cell division (reviewed in Ref. 15Blackburn E.H. Nature. 2000; 408: 53-56Google Scholar). Telomere-associated senescence can be bypassed in some cell lines by the expression of telomerase, which extends telomeres (14Campisi J. Kim S. Lim C.S. Rubio M. Exp. Gerontol. 2001; 36: 1619-1637Google Scholar). Evidence suggests that proper telomere function in mammalian cells is maintained by a nucleoprotein complex, together with structural features of the telomeric DNA. Telomere repeat binding factors TRF1 and TRF2 regulate telomere length, and TRF2 defects induce growth arrest and telomere end fusions (16Smogorzewska A. van Steensel B. Bianchi A. Oelmann S. Schaefer M.R. Schna